U.S. patent application number 16/714891 was filed with the patent office on 2020-04-16 for liposomes having useful n:p ratio for delivery of rna molecules.
This patent application is currently assigned to GLAXOSMITHKLINE BIOLOGICALS SA. The applicant listed for this patent is GLAXOSMITHKLINE BIOLOGICALS SA. Invention is credited to Andrew GEALL, Ayush VERMA.
Application Number | 20200113831 16/714891 |
Document ID | / |
Family ID | 46579331 |
Filed Date | 2020-04-16 |
United States Patent
Application |
20200113831 |
Kind Code |
A1 |
GEALL; Andrew ; et
al. |
April 16, 2020 |
LIPOSOMES HAVING USEFUL N:P RATIO FOR DELIVERY OF RNA MOLECULES
Abstract
Nucleic acid immunisation is achieved by delivering a RNA
encapsulated within a liposome comprising a cationic lipid, wherein
the liposome and the RNA have a N:P ratio of between 1:1 and
20:1.
Inventors: |
GEALL; Andrew; (Littleton,
MA) ; VERMA; Ayush; (Morrisville, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE BIOLOGICALS SA |
Rixensart |
|
BE |
|
|
Assignee: |
GLAXOSMITHKLINE BIOLOGICALS
SA
Rixensart
BE
|
Family ID: |
46579331 |
Appl. No.: |
16/714891 |
Filed: |
December 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14130748 |
Jan 3, 2014 |
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PCT/US2012/045829 |
Jul 6, 2012 |
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16714891 |
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61505088 |
Jul 6, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 39/39 20130101; C12N 2760/18534 20130101; A61K 9/1272
20130101; C12N 2770/36143 20130101; A61K 39/12 20130101; A61K
2039/53 20130101; A61K 39/155 20130101; A61K 9/127 20130101; A61K
2039/55555 20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/12 20060101 A61K039/12; A61K 39/155 20060101
A61K039/155; A61K 39/39 20060101 A61K039/39 |
Claims
1.-15. (canceled)
16. A method for raising a protective immune response in a
vertebrate, comprising the step of administering to the vertebrate
an effective amount of a liposome comprising a cationic lipid and
the liposome and RNA have a N:P ratio of between 1:1 and 20:1.
17. A method for raising a protective immune response in a
vertebrate, comprising the step of administering to the vertebrate
an effective amount of a pharmaceutical composition comprising a
liposome wherein the liposome comprises a cationic lipid and the
liposome and RNA have a N:P ratio of between 1:1 and 20:1.
18. The method of claim 16, wherein the vertebrate is a human or a
veterinary mammal.
19. The method of claim 16, wherein the RNA is
self-replicating.
20. The method of claim 19, wherein the self-replicating RNA
encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA
from the self-replicating RNA molecule and (ii) an immunogen.
21. The method of claim 19, wherein the self-replicating RNA has
two open reading frames, the first of which encodes an alphavirus
replicase and the second of which encodes the immunogen.
22. The method of claim 19, wherein the immunogen can elicit an
immune response in vivo against respiratory syncytial virus
glycoprotein F.
23. The method of claim 16, wherein the immunogen may elicit an
immune response against a bacterium, virus, fungus, or
parasite.
24. The method of claim 16, wherein the immune response may
comprise an antibody response.
25. The method of claim 16, wherein the immune response may
comprise a cell-mediated immune response.
Description
[0001] This application is a continuing application of U.S. patent
application Ser. No. 14/130,748, filed on Jan. 3, 2014, which is a
national stage entry of PCT/US2012/045829, filed Jul. 6, 2012 which
claims the benefit of U.S. Provisional No. 61/505,088 filed on Jul.
6, 2011. The entire contents of the foregoing application are
incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention is in the field of non-viral delivery of RNAs
for immunisation.
BACKGROUND ART
[0003] The delivery of nucleic acids for immunising animals has
been a goal for several years. Various approaches have been tested,
including the use of DNA or RNA, of viral or non-viral delivery
vehicles (or even no delivery vehicle, in a "naked" vaccine), of
replicating or non-replicating vectors, or of viral or non-viral
vectors.
[0004] There remains a need for further and improved nucleic acid
vaccines and, in particular, for improved ways of delivering
nucleic acid vaccines.
DISCLOSURE OF THE INVENTION
[0005] According to the invention, nucleic acid immunisation is
achieved by delivering a RNA encapsulated within a liposome
comprising a cationic lipid, wherein the liposome and the RNA have
a N:P ratio of between 1:1 and 20:1. The "N:P ratio" refers to the
molar ratio of nitrogen atoms in the cationic lipid to phosphates
in the RNA. The liposomes are useful for in vivo delivery of RNA to
a vertebrate cell.
[0006] Thus the invention provides a liposome in which a RNA is
encapsulated, wherein the liposome comprises a cationic lipid, the
RNA encodes an immunogen, and the liposome & RNA have a N:P
ratio of between 1:1 and 20:1.
[0007] The invention also provides a process for preparing a
liposome which encapsulates an immunogen-encoding RNA, wherein the
liposome is formed by mixing liposome-forming components with an
immunogen-encoding RNA, wherein the liposome-forming ingredients
comprise a cationic lipid, and wherein the cationic lipid and the
RNA are mixed at a N:P ratio of between 1:1 and 20:1.
[0008] The Liposome
[0009] Various amphiphilic lipids can form bilayers in an aqueous
environment to encapsulate a RNA-containing aqueous core as a
liposome. These lipids can have an anionic, cationic or
zwitterionic hydrophilic head group. Formation of liposomes from
anionic phospholipids dates back to the 1960s, and cationic
liposome-forming lipids have been studied since the 1990s. Some
phospholipids are anionic whereas other are zwitterionic and others
are cationic. Suitable classes of phospholipid include, but are not
limited to, phosphatidylethanolamines, phosphatidylcholines,
phosphatidylserines, and phosphatidyl-glycerols, and some useful
phospholipids are listed in Table 1. Useful cationic lipids
include, but are not limited to, dioleoyl trimethylammonium propane
(DOTAP), 1,2-distearyloxv-N,N-dimethyl-3-aminopropane (DSDMA),
1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA),
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),
1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA).
Zwitterionic lipids include, but are not limited to, acyl
zwitterionic lipids and ether zwitterionic lipids. Examples of
useful zwitterionic lipids are DPPC, DOPC and
dodecylphosphocholine. The lipids can be saturated or unsaturated.
The use of at least one unsaturated lipid for preparing liposomes
is preferred. If an unsaturated lipid has two tails, both tails can
be unsaturated, or it can have one saturated tail and one
unsaturated tail.
[0010] Liposomal particles of the invention can be formed from a
single lipid or from a mixture of lipids, provided that at least
one cationic lipid is used. Where a mixture is used, it may
comprise both saturated and unsaturated lipids. For example, a
mixture useful with the invention may comprise DlinDMA (cationic,
unsaturated), DSPC (zwitterionic, saturated), and/or DMG (anionic,
saturated). Where a mixture of lipids is used, not all of the
component lipids in the mixture need to be charged e.g. one or more
amphiphilic lipids, including a cationic lipid, can be mixed with
cholesterol.
[0011] Liposomal particles of the invention include at least one
cationic lipid whose head group includes at least one nitrogen atom
which is capable of being protonated. The number of these lipid
nitrogen atoms which can be protonated in the liposome contribute
to the "N:P ratio" used herein.
[0012] Preferred cationic lipids have a protonatable nitrogen with
a pKa in the range of 5.0 to 7.6. Ideally the lipid with a pKa in
this range has a tertiary amine; such lipids behave differently
from lipids such as DOTAP or DC-Chol, which have a quaternary amine
group. At physiological pH amines with a pKa in the range of 5.0 to
7.6 have neutral or reduced surface charge, whereas a lipid such as
DOTAP is strongly cationic. The inventors have found that liposomes
formed from quaternary amine lipids (e.g. DOTAP) are less suitable
for delivery of immunogen-encoding RNA than liposomes formed from
tertiary amine lipids (e.g. DLinDMA).
[0013] Within this pKa range, preferred lipids have a pKa of 5.5 to
6.7 e.g. between 5.6 and 6.8, between 5.6 and 6.3, between 5.6 and
6.0, between 5.5 and 6.2, or between 5.7 and 5.9. The pKa is the pH
at which 50% of the lipids are charged, lying halfway between the
point where the lipids are completely charged and the point where
the lipids are completely uncharged. It can be measured in various
ways, but is preferably measured using the method disclosed below
in the section entitled "pKa measurement". The pKa typically should
be measured for the lipid alone rather than for the lipid in the
context of a mixture which also includes other lipids (e.g. not as
performed in reference 5, which looks at the pKa of a SNALP rather
than of the individual lipids).
[0014] Preferred lipids with a pKa in this range have a tertiary
amine. For example, they may comprise
1,2-dilinoleyloxv-N,N-dimethyl-3-aminopropane (DLinDMA; pKa 5.8)
and/or 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA).
Another suitable lipid having a tertiary amine is
1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA). See ref. 1. Some
of the amino acid lipids of reference 2 may also be used, as can
certain of the amino lipids of reference 3. Further useful lipids
with tertiary amines in their headgroups are disclosed in reference
4, the complete contents of which are incorporated herein by
reference.
[0015] The hydrophilic portion of a lipid can be PEGylated (i.e.
modified by covalent attachment of a polyethylene glycol). This
modification can increase stability and prevent non-specific
adsorption of the liposomes. For instance, lipids can be conjugated
to PEG using techniques such as those disclosed in reference 1 and
5. Various lengths of PEG can be used e.g. between 0.5-8 kDa.
[0016] Where a liposome is formed from a mixture of lipids, it is
preferred that the proportion of those lipids which have a
protonatable nitrogen should be between 20-80% of the total amount
of lipids e.g. between 30-70%, or between 40-60%. The remainder of
the lipid content can be made of e.g. cholesterol (e.g. 35-50%
cholesterol) and/or DMG (optionally PEGylated) and/or DSPC. Such
mixtures are used below. These % values are mole percentages. A
mixture of DSPC, DlinDMA, PEG-DMG and cholesterol is used in the
examples.
[0017] Liposomal particles are usually divided into three groups:
multilamellar vesicles (MLV); small unilamellar vesicles (SUV); and
large unilamellar vesicles (LUV). MLVs have multiple bilayers in
each vesicle, forming several separate aqueous compartments. SUVs
and LUVs have a single bilayer encapsulating an aqueous core; SUVs
typically have a diameter .ltoreq.50 nm, and LUVs have a diameter
>50 nm. Liposomal particles of the invention are ideally LUVs
with a diameter in the range of 50-220 nm. For a composition
comprising a population of LUVs with different diameters: (i) at
least 80% by number should have diameters in the range of 20-220
nm, (ii) the average diameter (Zav, by intensity) of the population
is ideally in the range of 40-200 nm, and/or (iii) the diameters
should have a polydispersity index <0.2.
[0018] The lipid DLinDMA is sometimes referred to herein as RV01;
this is a preferred cationic lipid for use with the invention,
although in some embodiments the liposomes does not comprise
DLinDMA:
##STR00001##
[0019] Another useful lipid, disclosed in reference 4, is sometimes
referred to herein as RV05:
##STR00002##
[0020] The lipid DOTAP is sometimes referred to herein as RV13, but
it is not a preferred lipid.
[0021] Mixing Process
[0022] Techniques for preparing suitable liposomes are well known
in the art e.g. see references 6 to 8. One useful method is
described in reference 9 and involves mixing (i) an ethanolic
solution of the lipids (ii) an aqueous solution of the nucleic acid
and (iii) buffer, followed by mixing, equilibration, dilution and
purification. Preferred liposomes of the invention are obtainable
by this mixing process.
[0023] A useful process for preparing a RNA-containing liposome
comprises steps of (a) mixing RNA with a lipid at a pH which is
below the lipid's pKa but is above 4.5; then (b) increasing the pH
to be above the lipid's pKa. Thus a cationic lipid is positively
charged during liposome formation in step (a), but the pH change
thereafter means that the majority (or all) of the positively
charged groups become neutral. This process is advantageous for
preparing liposomes of the invention, and by avoiding a pH below
4.5 during step (a) the stability of the encapsulated RNA is
improved.
[0024] The pH in step (a) is above 4.5, and is ideally above 4.8.
Using a pH in the range of 5.0 to 6.0, or in the range of 5.0 to
5.5, can provide suitable liposomes.
[0025] The increased pH in step (b) is above the lipid's pKa. The
pH is ideally increased to a pH less than 9, and preferably less
than 8. Depending on the lipid's pKa, the pH in step (b) may thus
be increased to be within the range of 6 to 8 e.g. to pH
6.5.+-.0.3. The pH increase of step (b) can be achieved by
transferring the liposomes into a suitable buffer e.g. into
phosphate-buffered saline. The pH increase of step (b) is ideally
performed after liposome formation has taken place.
[0026] RNA used in step (a) can be in aqueous solution, for mixing
with an organic solution of the lipid (e.g. an ethanolic solution,
as in ref. 9). The mixture can then be diluted to form liposomes,
after which the pH can be increased in step (b).
[0027] The RNA
[0028] Liposomes of the invention include a RNA molecule which
(unlike siRNA) encodes an immunogen. After in vivo administration
of the liposomes, RNA is released and is translated inside a cell
to provide the immunogen min situ.
[0029] The RNA is +-stranded, and so it can be translated without
needing any intervening replication steps such as reverse
transcription. Preferred +-stranded RNAs are self-replicating,
unlike reference 10. A self-replicating RNA molecule (replicon)
can, when delivered to a cell even without any proteins, lead to
the production of multiple daughter RNAs by transcription from
itself (via an antisense copy which it generates from itself). A
self-replicating RNA molecule is thus typically a +-strand molecule
which can be directly translated after delivery to a cell, and this
translation provides a RNA-dependent RNA polymerase which then
produces both antisense and sense transcripts from the delivered
RNA. Thus the delivered RNA leads to the production of multiple
daughter RNAs. These daughter RNAs, as well as collinear subgenomic
transcripts, may be translated themselves to provide in situ
expression of an encoded immunogen, or may be transcribed to
provide further transcripts with the same sense as the delivered
RNA which are translated to provide in situ expression of the
immunogen. The overall results of this sequence of transcriptions
is a huge amplification in the number of the introduced replicon
RNAs and so the encoded immunogen becomes a major polypeptide
product of the cells.
[0030] One suitable system for achieving self-replication in this
manner is to use an alphavirus-based replicon. These replicons are
+-stranded RNAs which lead to translation of a replicase (or
replicase-transcriptase) after delivery to a cell. The replicase is
translated as a polyprotein which auto-cleaves to provide a
replication complex which creates genomic --strand copies of the
+-strand delivered RNA. These --strand transcripts can themselves
be transcribed to give further copies of the +-stranded parent RNA
and also to give a subgenomic transcript which encodes the
immunogen. Translation of the subgenomic transcript thus leads to
in situ expression of the immunogen by the infected cell. Suitable
alphavirus replicons can use a replicase from a Sindbis virus, a
Semliki forest virus, an eastern equine encephalitis virus, a
Venezuelan equine encephalitis virus, etc. Mutant or wild-type
virus sequences can be used e.g. the attenuated TC83 mutant of VEEV
has been used in replicons [11].
[0031] A preferred self-replicating RNA molecule thus encodes (i) a
RNA-dependent RNA polymerase which can transcribe RNA from the
self-replicating RNA molecule and (ii) an immunogen. The polymerase
can be an alphavirus replicase e.g. comprising one or more of
alphavirus proteins nsP1, nsP2, nsP3 and nsP4.
[0032] Whereas natural alphavirus genomes encode structural virion
proteins in addition to the non-structural replicase polyprotein,
it is preferred that the self-replicating RNA molecules of the
invention do not encode alphavirus structural proteins. Thus a
preferred self-replicating RNA can lead to the production of
genomic RNA copies of itself in a cell, but not to the production
of RNA-containing virions. The inability to produce these virions
means that, unlike a wild-type alphavirus, the self-replicating RNA
molecule cannot perpetuate itself in infectious form. The
alphavirus structural proteins which are necessary for perpetuation
in wild-type viruses are absent from self-replicating RNAs of the
invention and their place is taken by gene(s) encoding the
immunogen of interest, such that the subgenomic transcript encodes
the immunogen rather than the structural alphavirus virion
proteins.
[0033] Thus a self-replicating RNA molecule useful with the
invention may have two open reading frames. The first (5') open
reading frame encodes a replicase; the second (3') open reading
frame encodes an immunogen. In some embodiments the RNA may have
additional (e.g. downstream) open reading frames e.g. to encode
further immunogens (see below) or to encode accessory
polypeptides.
[0034] A preferred self-replicating RNA molecule has a 5' cap (e.g.
a 7-methylguanosine). This cap can enhance in vivo translation of
the RNA. In some embodiments the 5' sequence of the
self-replicating RNA molecule must be selected to ensure
compatibility with the encoded replicase.
[0035] A self-replicating RNA molecule may have a 3' poly-A tail.
It may also include a poly-A polymerase recognition sequence (e.g.
AAUAAA) near its 3' end.
[0036] Self-replicating RNA molecules can have various lengths but
they are typically 5000-25000 nucleotides long e.g. 8000-15000
nucleotides, or 9000-12000 nucleotides. Thus the RNA is longer than
seen in siRNA delivery.
[0037] Self-replicating RNA molecules will typically be
single-stranded. Single-stranded RNAs can generally initiate an
adjuvant effect by binding to TLR7, TLR8, RNA helicases and/or PKR.
RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and
this receptor can also be triggered by dsRNA which is formed either
during replication of a single-stranded RNA or within the secondary
structure of a single-stranded RNA.
[0038] The self-replicating RNA can conveniently be prepared by in
vitro transcription (IVT). IVT can use a (cDNA) template created
and propagated in plasmid form in bacteria, or created
synthetically (for example by gene synthesis and/or polymerase
chain-reaction (PCR) engineering methods). For instance, a
DNA-dependent RNA polymerase (such as the bacteriophage T7, T3 or
SP6 RNA polymerases) can be used to transcribe the self-replicating
RNA from a DNA template. Appropriate capping and poly-A addition
reactions can be used as required (although the replicon's poly-A
is usually encoded within the DNA template). These RNA polymerases
can have stringent requirements for the transcribed 5'
nucleotide(s) and in some embodiments these requirements must be
matched with the requirements of the encoded replicase, to ensure
that the IVT-transcribed RNA can function efficiently as a
substrate for its self-encoded replicase.
[0039] As discussed in reference 12, the self-replicating RNA can
include (in addition to any 5' cap structure) one or more
nucleotides having a modified nucleobase. For instance, a
self-replicating RNA can include one or more modified pyrimidine
nucleobases, such as pseudouridine and/or 5-methylcytosine
residues. In some embodiments, however, the RNA includes no
modified nucleobases, and may include no modified nucleotides i.e.
all of the nucleotides in the RNA are standard A, C, G and U
ribonucleotides (except for any 5' cap structure, which may include
a 7'-methylguanosine). In other embodiments, the RNA may include a
5' cap comprising a 7'-methylguanosine, and the first 1, 2 or 3 5'
ribonucleotides may be methylated at the 2' position of the
ribose.
[0040] A RNA used with the invention ideally includes only
phosphodiester linkages between nucleosides, but in some
embodiments it can contain phosphoramidate, phosphorothioate,
and/or methylphosphonate linkages.
[0041] Ideally, a liposome includes fewer than 10 different species
of RNA e.g. 5, 4, 3, or 2 different species, most preferably, a
liposome includes a single RNA species i.e. all RNA molecules in
the liposome have the same sequence and same length.
[0042] The N:P Ratio
[0043] The invention uses liposomes prepared to have a N:P ratio of
between 1:1 and 20:1. As mentioned above, the "N:P ratio" refers to
the molar ratio of protonatable nitrogen atoms in the liposome's
cationic lipids (typically solely in the lipid's headgroup) to
phosphates in the RNA.
[0044] Useful N:P ratios are from about 2:1 to about 18:1, from
about 4:1 to 16:1, from about 6:1 to about 14:1, from about 8:1 to
about 12:1. Preferred N:P ratios are between 3:1 and 11:1. Useful
N:P ratios as exemplified below include 2:1, 4:1, 8:1 and 10:1.
[0045] As shown below, the N:P ratio can have an impact on
immunogenicity, with lower ratios being preferred e.g. lower than
8:1, lower than 7:1, lower than 6:1, lower than 5:1, or <4:1
e.g. between 2:1 and 4:1 inclusive.
[0046] In some embodiments the N:P ratio is not between 7:1 and
9:1. In some embodiments the N:P ratio is not between 7.5:1 and
8.5:1. In some embodiments the N:P ratio is not 8:1.
[0047] The N:P ratio can be modified by varying the proportions of
cationic lipid and RNA during liposome formation. This is achieved
most conveniently by modifying the RNA concentration in aqueous
material which is used during formation of liposomes from a
suitable combination of liposome-forming components. For example,
in the method mentioned above the ethanolic solution of
liposome-forming lipids can be kept constant while the
concentration of RNA in the aqueous solution is varied. A higher
RNA concentration will decrease the N:P ratio, whereas a lower RNA
concentration will increase the N:P ratio.
[0048] For certain purposes the N:P ratio can be based on
assumptions. For instance, if .mu.g of a RNA molecule is assumed to
contain 3 nmol of anionic phosphate, and each .mu.g of DlinDMA is
assumed to contains 1.6 nmol of cationic nitrogen, the calculation
can be simplified for convenience.
[0049] The Immunogen
[0050] RNA molecules used with the invention encode a polypeptide
immunogen. After administration of the liposomes the immunogen is
translated in vivo and can elicit an immune response in the
recipient. The immunogen may elicit an immune response against a
bacterium, a virus, a fungus or a parasite (or, in some
embodiments, against an allergen; and in other embodiments, against
a tumor antigen). The immune response may comprise an antibody
response (usually including IgG) and/or a cell-mediated immune
response. The polypeptide immunogen will typically elicit an immune
response which recognises the corresponding bacterial, viral,
fungal or parasite (or allergen or tumour) polypeptide, but in some
embodiments the polypeptide may act as a mimotope to elicit an
immune response which recognises a bacterial, viral, fungal or
parasite saccharide. The immunogen will typically be a surface
polypeptide e.g. an adhesin, a hemagglutinin, an envelope
glycoprotein, a spike glycoprotein, etc.
[0051] RNA molecules can encode a single polypeptide immunogen or
multiple polypeptides. Multiple immunogens can be presented as a
single polypeptide immunogen (fusion polypeptide) or as separate
polypeptides. If immunogens are expressed as separate polypeptides
then one or more of these may be provided with an upstream IRES or
an additional viral promoter element. Alternatively, multiple
immunogens may be expressed from a polyprotein that encodes
individual immunogens fused to a short autocatalytic protease (e.g.
foot-and-mouth disease virus 2A protein), or as inteins.
[0052] The RNA encodes an immunogen. For the avoidance of doubt,
the invention does not encompass RNA which encodes a firefly
luciferase or which encodes a fusion protein of E. coli
.beta.-galactosidase or which encodes a green fluorescent protein
(GFP). Also, the RNA is not total mouse thymus RNA. Also, the
invention does not encompass RNA which encodes a secreted alkaline
phosphatase.
[0053] In some embodiments the immunogen elicits an immune response
against one of these bacteria: [0054] Neisseria meningitidis:
useful immunogens include, but are not limited to, membrane
proteins such as adhesins, autotransporters, toxins, iron
acquisition proteins, and factor H binding protein. A combination
of three useful polypeptides is disclosed in reference 13. [0055]
Streptococcus pneumoniae: useful polypeptide immunogens are
disclosed in reference 14. These include, but are not limited to,
the RrgB pilus subunit, the beta-N-acetyl-hexosaminidase precursor
(spr0057), spr0096. General stress protein GSP-781 (spr2021,
SP2216), serine/threonine kinase StkP (SP1732), and pneumococcal
surface adhesin PsaA. [0056] Streptococcus pyogenes: useful
immunogens include, but are not limited to, the polypeptides
disclosed in references 15 and 16. [0057] Moraxella catarrhalis.
[0058] Bordetella pertussis: Useful pertussis immunogens include,
but are not limited to, pertussis toxin or toxoid (PT), filamentous
haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3. [0059]
Staphylococcus aureus: Useful immunogens include, but are not
limited to, the polypeptides disclosed in reference 17, such as a
hemolysin, esxA, esxB, ferrichrome-binding protein (sta006) and/or
the sta011 lipoprotein. [0060] Clostridium tetani: the typical
immunogen is tetanus toxoid. [0061] Cornynebacterium diphtheriae:
the typical immunogen is diphtheria toxoid. [0062] Haemophilus
influenzae: Useful immunogens include, but are not limited to, the
polypeptides disclosed in references 18 and 19. [0063] Pseudomonas
aeruginosa [0064] Streptococcus agalactiae: useful immunogens
include, but are not limited to, the polypeptides disclosed in
reference 15. [0065] Chlamydia trachomatis: Useful immunogens
include, but are not limited to, PepA, LcrE, ArtJ, DnaK, CT398,
OmpH-like, L7/L12, OmcA, AtoS, CT547, Eno, HtrA and MurG (e.g. as
disclosed in reference 20. LcrE [21] and HtrA [22] are two
preferred immunogens. [0066] Chlamydia pneumoniae: Useful
immunogens include, but are not limited to, the polypeptides
disclosed in reference 23. [0067] Helicobacter pylori: Useful
immunogens include, but are not limited to, CagA, VacA, NAP, and/or
urease [24]. [0068] Escherichia coli: Useful immunogens include,
but are not limited to, immunogens derived from enterotoxigenic E.
coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering
E. coli (DAEC), enteropathogenic E. coli (EPEC), extraintestinal
pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli (EHEC).
ExPEC strains include uropathogenic E. coli (UPEC) and
meningitis/sepsis-associated E. coli (MNEC). Useful UPEC
polypeptide immunogens are disclosed in references 25 and 26.
Useful MNEC immunogens are disclosed in reference 27. A useful
immunogen for several E. coli types is AcfD [28]. [0069] Bacillus
anthracis [0070] Yersinia pestis: Useful immunogens include, but
are not limited to, those disclosed in references 29 and 30. [0071]
Staphylococcus epidermis [0072] Clostridium perfringens or
Clostridium botulinums [0073] Legionella pneumophila [0074]
Coxiella burnetti [0075] Brucella, such as B. abortus, B. canis, B.
melitensis, B. neotomae, B. ovis, B. suis, B. pinnipediae. [0076]
Francisella, such as F. novicida, F. philomiragia, F. tularensis.
[0077] Neisseria gonorrhoeae [0078] Treponema pallidum [0079]
Haemophilus ducreyi [0080] Enterococcus faecalis or Enterococcus
faecium [0081] Staphylococcus saprophyticus [0082] Yersinia
enterocolitica [0083] Mycobacterium tuberculosis [0084] Rickettsia
[0085] Listeria monocytogenes [0086] Vibrio cholerae [0087]
Salmonella typhi [0088] Borrelia burgdorferi [0089] Porphyromonas
gingivalis [0090] Klebsiella
[0091] In some embodiments the immunogen elicits an immune response
against one of these viruses: [0092] Orthomyxovirus: Useful
immunogens can be from an influenza A, B or C virus, such as the
hemagglutinin, neuraminidase or matrix M2 proteins. Where the
immunogen is an influenza A virus hemagglutinin it may be from any
subtype e.g. H1, H2. H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,
H13, H14, H15 or H16. [0093] Paramnxoviridae viruses: Viral
immunogens include, but are not limited to, those derived from
Pneumoviruses (e.g. respiratory syncytial virus, RSV),
Rubulaviruses (e.g. mumps virus), Paramyxoviruses (e.g.
parainfluenza virus), Metapneumoviruses and Morbilliviruses (e.g.
measles). In some embodiments, however, the immunogen is not a RSV
protein. [0094] Poxviridae: Viral immunogens include, but are not
limited to, those derived from Orthopoxvirus such as Variola vera,
including but not limited to, Variola major and Variola minor.
[0095] Picornavirus: Viral immunogens include, but are not limited
to, those derived from Picornaviruses, such as Enteroviruses,
Rhinoviruses, Heparnavirus, Cardioviruses and Aphthoviruses. In one
embodiment, the enterovirus is a poliovirus e.g. a type 1, type 2
and/or type 3 poliovirus. In another embodiment, the enterovirus is
an EV71 enterovirus. In another embodiment, the enterovirus is a
coxsackie A or B virus. [0096] Bunyavirus: Viral immunogens
include, but are not limited to, those derived from an
Orthobunyavirus, such as California encephalitis virus, a
Phlebovirus, such as Rift Valley Fever virus, or a Nairovirus, such
as Crimean-Congo hemorrhagic fever virus. [0097] Heparnavirus:
Viral immunogens include, but are not limited to, those derived
from a Heparnavirus, such as hepatitis A virus (HAV). [0098]
Filovirus: Viral immunogens include, but are not limited to, those
derived from a filovirus, such as an Ebola virus (including a
Zaire, Ivory Coast, Reston or Sudan ebolavirus) or a Marburg virus.
[0099] Togavirus: Viral immunogens include, but are not limited to,
those derived from a Togavirus, such as a Rubivirus, an Alphavirus,
or an Arterivirus. This includes rubella virus. [0100] Flavivirus:
Viral immunogens include, but are not limited to, those derived
from a Flavivirus, such as Tick-borne encephalitis (TBE) virus,
Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese
encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis
virus, St. [0101] Louis encephalitis virus, Russian spring-summer
encephalitis virus, Powassan encephalitis virus. [0102] Pestivirus:
Viral immunogens include, but are not limited to, those derived
from a Pestivirus, such as Bovine viral diarrhea (BVDV), Classical
swine fever (CSFV) or Border disease (BDV). [0103] Hepadnavirus:
Viral immunogens include, but are not limited to, those derived
from a Hepadnavirus, such as Hepatitis B virus. A composition can
include hepatitis B virus surface antigen (HBsAg). [0104] Other
hepatitis viruses: A composition can include an immunogen from a
hepatitis C virus, delta hepatitis virus, hepatitis E virus, or
hepatitis G virus. [0105] Rhabdovirus: Viral immunogens include,
but are not limited to, those derived from a Rhabdovirus, such as a
Lyssavirus (e.g. a Rabies virus) and Vesiculovirus (VSV). [0106]
Caliciviridae: Viral immunogens include, but are not limited to,
those derived from Calciviridae, such as Norwalk virus (Norovirus),
and Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain
Virus. [0107] Coronavirus: Viral immunogens include, but are not
limited to, those derived from a SARS coronavirus, avian infectious
bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine
transmissible gastroenteritis virus (TGEV). The coronavirus
immunogen may be a spike polypeptide. [0108] Retrovirus: Viral
immunogens include, but are not limited to, those derived from an
Oncovirus, a Lentivirus (e.g. HIV-1 or HIV-2) or a Spumavirus.
[0109] Reovirus: Viral immunogens include, but are not limited to,
those derived from an Orthoreovirus, a Rotavirus, an Orbivirus, or
a Coltivirus. [0110] Parvovirus: Viral immunogens include, but are
not limited to, those derived from Parvovirus B19. [0111]
Herpesvirus: Viral immunogens include, but are not limited to,
those derived from a human herpesvirus, such as, by way of example
only, Herpes Simplex Viruses (HSV) (e.g. HSV types 1 and 2),
Varicella-zoster virus (VZV), Epstein-Barr virus (EBV),
Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human
Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8). [0112]
Papovaviruses: Viral immunogens include, but are not limited to,
those derived from Papillomaviruses and Polyomaviruses. The (human)
papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18,
31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65 e.g. from one or
more of serotypes 6, 11, 16 and/or 18. [0113] Adenovinrs: Viral
immunogens include those derived from adenovirus serotype 36
(Ad-36).
[0114] In some embodiments, the immunogen elicits an immune
response against a virus which infects fish, such as: infectious
salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV),
infectious pancreatic necrosis virus (IPNV), channel catfish virus
(CCV), fish lymphocystis disease virus (FLDV), infectious
hematopoietic necrosis virus (IHNV), koi herpesvirus, salmon
picorna-like virus (also known as picorna-like virus of atlantic
salmon), landlocked salmon virus (LSV), atlantic salmon rotavirus
(ASR), trout strawberry disease virus (TSD), coho salmon tumor
virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).
[0115] Fungal immunogens may be derived from Dermatophytres,
including: Epidermophyton floccusum, Microsporum audouni.
Microsporum canis. Microsporum distortum. Microsporum equinum.
Microsporum gypsum. Microsporum nanum. Trichophyton concentricum.
Trichophyton equinum. Trichophyton gallinae. Trichophyton gypseum.
Trichophyton megnini. Trichophyton mentagrophytes, Trichophyton
quinckeanum. Trichophyton rubrum, Trichophyton schoenleini.
Trichophyton tonsurans. Trichophyton verrucosum. T. verrucosum var.
album, var. discoides, var. ochraccum, Trichophyton violaceum,
and/or Trichophyton faviforme; or from Aspergillus fumigatus.
Aspergillus flavus. Aspergillus niger. Aspergillus nidulans,
Aspergillus terreus, Aspergillus sydowii. Aspergillus flavatus.
Aspergillus glaucus. Blastoschizomyces capitatus. Candida albicans.
Candida enolase. Candida tropicalis. Candida glabrata. Candida
krusei. Candida parapsilosis. Candida stellatoidea. Candida kusei.
Candida parakwsei. Candida lusitaniae. Candida pseudotropicalis,
Candida guilliermondi. Cladosporium carrionmi. Coccidioides
immitis. Blastomyces dermatidis, Cryptococcus neoformans,
Geotrichum clavatum. Histoplasma capsulatum. Klebsiella pneumoniae,
Microsporidia, Encephalitozoon spp., Septata intestinalis and
Enterocytozoon bieneusi: the less common are Brachiola spp,
Microsporidium spp., Nosema spp., Pleistophora spp.,
Trachipleistophora spp., Vittaforma spp Paracoccidioides
brasiliensis, Pneumocystis carinii, Pythiumn insidiosum,
Pityrosporun ovale, Sacharomyces cerevisae, Saccharomyces
boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix
schencki, Trichosporon beigelti, Toxoplasma gondii, Penicillium
marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp.,
Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus
spp, Mucor spp, Absidia spp. Mortierella spp. Cunninghamella spp.
Saksenaea spp., Alternaria spp. Curvularia spp. Helminthosporium
spp. Fusarium spp. Aspergillus spp. Penicillium spp. Monolinia spp.
Rhizoctonia spp. Paecilomyces spp. Pithomyces spp. and Cladosporium
spp.
[0116] In some embodiments the immunogen elicits an immune response
against a parasite from the Plasmodium genus, such as P.
falciparum, P. vivax, P. malarinae or P. ovale. Thus the invention
may be used for immunising against malaria. In some embodiments the
immunogen elicits an immune response against a parasite from the
Caligidae family, particularly those from the Lepeophtheirus and
Caligus genera e.g. sea lice such as Lepeophtheirus salmonis or
Caligus rogercresseyi.
[0117] In some embodiments the immunogen elicits an immune response
against: pollen allergens (tree-, herb, weed-, and grass pollen
allergens): insect or arachnid allergens (inhalant, saliva and
venom allergens, e.g. mite allergens, cockroach and midges
allergens, hymenopthera venom allergens); animal hair and dandruff
allergens (from e.g. dog, cat, horse, rat, mouse, etc.); and food
allergens (e.g. a gliadin). Important pollen allergens from trees,
grasses and herbs are such originating from the taxonomic orders of
Fagales, Oleales, Pinales and platanaceae including, but not
limited to, birch (Betula), alder (Alnus), hazel (Corylus),
hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and
Juniperus), plane tree (Platanus), the order of Poales including
grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis,
Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and
Urticales including herbs of the genera Ambrosia, Artemisia, and
Parietaria. Other important inhalation allergens are those from
house dust mites of the genus Dermatophagoides and Euroglyphus,
storage mite e.g. Lepidoglyphys, Glycyphagus and Tyrophagus, those
from cockroaches, midges and fleas e.g. Blatella, Periplaneta,
Chironomus and Ctenocepphalides, and those from mammals such as
cat, dog and horse, venom allergens including such originating from
stinging or biting insects such as those from the taxonomic order
of Hymenoptera including bees (Apidae), wasps (Vespidea), and ants
(Formicoidae).
[0118] In some embodiments the immunogen is a tumor antigen
selected from: (a) cancer-testis antigens such as NY-ESO-1, SSX2,
SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for
example, GAGE-1, GAGE-2, MAGE-1, MAGE-2. MAGE-3, MAGE-4, MAGE-5,
MAGE-6, and MAGE-12 (which can be used, for example, to address
melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and
bladder tumors: (b) mutated antigens, for example, p53 (associated
with various solid tumors, e.g., colorectal, lung, head and neck
cancer), p21/Ras (associated with, e.g., melanoma, pancreatic
cancer and colorectal cancer), CDK4 (associated with, e.g.,
melanoma), MUMI (associated with, e.g., melanoma), caspase-8
(associated with, e.g., head and neck cancer), CIA 0205 (associated
with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated
with, e.g., melanoma), TCR (associated with, e.g., T-cell
non-Hodgkins lymphoma), BCR-abl (associated with, e.g., chronic
myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27,
and LDLR-FUT; (c) over-expressed antigens, for example, Galectin 4
(associated with, e.g., colorectal cancer), Galectin 9 (associated
with, e.g., Hodgkin's disease), proteinase 3 (associated with,
e.g., chronic myelogenous leukemia), WT 1 (associated with, e.g.,
various leukemias), carbonic anhydrase (associated with, e.g.,
renal cancer), aldolase A (associated with, e.g., lung cancer),
PRAME (associated with, e.g., melanoma), HER-2/neu (associated
with, e.g., breast, colon, lung and ovarian cancer), mammaglobin,
alpha-fetoprotein (associated with, e.g., hepatoma), KSA
(associated with, e.g., colorectal cancer), gastrin (associated
with, e.g., pancreatic and gastric cancer), telomerase catalytic
protein, MUC-1 (associated with, e.g., breast and ovarian cancer),
G-250 (associated with, e.g, renal cell carcinoma), p53 (associated
with, e.g., breast, colon cancer), and carcinoembryonic antigen
(associated with, e.g., breast cancer, lung cancer, and cancers of
the gastrointestinal tract such as colorectal cancer); (d) shared
antigens, for example, melanoma-melanocyte differentiation antigens
such as MART-1/Melan A, gp100, MCIR, melanocyte-stimulating hormone
receptor, tyrosinase, tyrosinase related protein-1/TRP1 and
tyrosinase related protein-2/TRP2 (associated with, e.g.,
melanoma); (e) prostate associated antigens such as PAP. PSA. PSMA,
PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate cancer; (f)
immunoglobulin idiotypes (associated with myeloma and B cell
lymphomas, for example). In certain embodiments, tumor immunogens
include, but are not limited to, p15. Hom/Mel-40, H-Ras, E2A-PRL,
H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human
papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and
C virus antigens, human T-cell lymphotropic virus antigens,
TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9,
CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4,
791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA),
CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733
(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18. NB/70K, NY-CO-1,
RCAS1, SDCCAG16. TA-90 (Mac-2 binding protein/cyclophilin
C-associated protein), TAAL6, TAG72, TLP, TPS, and the like.
[0119] Pharmaceutical Compositions
[0120] Liposomes of the invention are useful as components in
pharmaceutical compositions for immunising subjects against various
diseases. These compositions will typically include a
pharmaceutically acceptable carrier in addition to the liposomes. A
thorough discussion of pharmaceutically acceptable carriers is
available in reference 31.
[0121] A pharmaceutical composition of the invention may include
one or more small molecule immunopotentiators. For example, the
composition may include a TLR2 agonist (e.g. Pam3CSK4), a TLR4
agonist (e.g. an aminoalkyl glucosaminide phosphate, such as
E6020), a TLR7 agonist (e.g. imiquimod), a TLR8 agonist (e.g.
resiquimod) and/or a TLR9 agonist (e.g. IC31). Any such agonist
ideally has a molecular weight of <2000 Da. In some embodiments
such agonist(s) are also encapsulated in the liposome (e.g.
together with the RNA), but in other embodiments they are
unencapsulated.
[0122] Pharmaceutical compositions of the invention may include the
particles in plain water (e.g. w.f.i.) or in a buffer e.g. a
phosphate buffer, a Tris buffer, a borate buffer, a succinate
buffer, a histidine buffer, or a citrate buffer. Buffer salts will
typically be included in the 5-20 mM range.
[0123] Pharmaceutical compositions of the invention may have a pH
between 5.0 and 9.5 e.g. between 6.0 and 8.0.
[0124] Compositions of the invention may include sodium salts (e.g.
sodium chloride) to give tonicity. A concentration of 10.+-.2 mg/ml
NaCl is typical e.g. about 9 mg/ml.
[0125] Compositions of the invention may include metal ion
chelators. These can prolong RNA stability by removing ions which
can accelerate phosphodiester hydrolysis. Thus a composition may
include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc. Such
chelators are typically present at between 10-500 .mu.M e.g. 0.1
mM. A citrate salt, such as sodium citrate, can also act as a
chelator, while advantageously also providing buffering
activity.
[0126] Pharmaceutical compositions of the invention may have an
osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between
240-360 mOsm/kg, or between 290-310 mOsm/kg.
[0127] Pharmaceutical compositions of the invention may include one
or more preservatives, such as thiomersal or 2-phenoxyethanol.
Mercury-free compositions are preferred, and preservative-free
vaccines can be prepared.
[0128] Pharmaceutical compositions of the invention are preferably
sterile.
[0129] Pharmaceutical compositions of the invention are preferably
non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard
measure) per dose, and preferably <0.1 EU per dose.
[0130] Pharmaceutical compositions of the invention are preferably
gluten free.
[0131] Pharmaceutical compositions of the invention may be prepared
in unit dose form. In some embodiments a unit dose may have a
volume of between 0.1-1.0 ml e.g. about 0.5 ml.
[0132] The compositions may be prepared as injectables, either as
solutions or suspensions. The composition may be prepared for
pulmonary administration e.g. by an inhaler, using a fine spray.
The composition may be prepared for nasal, aural or ocular
administration e.g. as spray or drops. Injectables for
intramuscular administration are typical.
[0133] Compositions comprise an immunologically effective amount of
particles, as well as any other components, as needed. By
`immunologically effective amount`, it is meant that the
administration of that amount to an individual, either in a single
dose or as part of a series, is effective for treatment or
prevention. This amount varies depending upon the health and
physical condition of the individual to be treated, age, the
taxonomic group of individual to be treated (e.g. non-human
primate, primate, etc.), the capacity of the individual's immune
system to synthesise antibodies, the degree of protection desired,
the formulation of the vaccine, the treating doctor's assessment of
the medical situation, and other relevant factors. It is expected
that the amount will fall in a relatively broad range that can be
determined through routine trials. The particle and RNA content of
compositions of the invention will generally be expressed in terms
of the amount of RNA per dose. A preferred dose has .ltoreq.100
.mu.g RNA (e.g. from 10-100 .mu.g, such as about 10 .mu.g, 25
.mu.g, 50 .mu.g, 75 .mu.g or 1001 .mu.g), but expression can be
seen even at lower levels.
[0134] The invention also provides a delivery device (e.g. syringe,
nebuliser, sprayer, inhaler, dermal patch, etc.) containing a
pharmaceutical composition of the invention. This device can be
used to administer the composition to a vertebrate subject.
[0135] Particles of the invention do not include ribosomes.
[0136] Methods of Treatment and Medical Uses
[0137] Liposomes and pharmaceutical compositions of the invention
are for in vivo use for eliciting an immune response against an
immunogen of interest.
[0138] The invention provides a method for raising an immune
response in a vertebrate comprising the step of administering an
effective amount of a liposome or pharmaceutical composition of the
invention. The immune response is preferably protective and
preferably involves antibodies and/or cell-mediated immunity. The
method may raise a booster response.
[0139] The invention also provides a liposome or pharmaceutical
composition of the invention for use in a method for raising an
immune response in a vertebrate.
[0140] The invention also provides the use of a liposome of the
invention in the manufacture of a medicament for raising an immune
response in a vertebrate.
[0141] By raising an immune response in the vertebrate by these
uses and methods, the vertebrate can be protected against various
diseases and/or infections e.g. against bacterial and/or viral
diseases as discussed above. The particles and compositions are
immunogenic, and are more preferably vaccine compositions. Vaccines
according to the invention may either be prophylactic (i.e. to
prevent infection) or therapeutic (i.e. to treat infection), but
will typically be prophylactic.
[0142] The vertebrate is preferably a mammal, such as a human or a
veterinary mammal (e.g. small animals, such as dogs or cats; or
large animals, such as horses, cattle, deer, goats, pigs): in some
embodiments, the vertebrate is not a mouse or a cotton rat or a
cow. Where the vaccine is for prophylactic use, the human is
preferably a child (e.g. a toddler or infant) or a teenager: where
the vaccine is for therapeutic use, the human is preferably a
teenager or an adult. A vaccine intended for children may also be
administered to adults e.g. to assess safety, dosage,
immunogenicity, etc.
[0143] Vaccines prepared according to the invention may be used to
treat both children and adults. Thus a human patient may be less
than 1 year old, less than 5 years old, 1-5 years old, 5-15 years
old, 15-55 years old, or at least 55 years old. Preferred patients
for receiving the vaccines are the elderly (e.g. .gtoreq.50 years
old, .gtoreq.60 years old, and preferably .gtoreq.65 years), the
young (e.g. .ltoreq.5 years old), hospitalised patients, healthcare
workers, armed service and military personnel, pregnant women, the
chronically ill, or immunodeficient patients. The vaccines are not
suitable solely for these groups, however, and may be used more
generally in a population.
[0144] Compositions of the invention will generally be administered
directly to a patient. Direct delivery may be accomplished by
parenteral injection (e.g. subcutaneously, intraperitoneally,
intravenously, intramuscularly, intradermally, or to the
interstitial space of a tissue). Alternative delivery routes
include rectal, oral (e.g. tablet, spray), buccal, sublingual,
vaginal, topical, transdermal or transcutaneous, intranasal,
ocular, aural, pulmonary or other mucosal administration.
Intradermal and intramuscular administration are two preferred
routes. Injection may be via a needle (e.g. a hypodermic needle),
but needle-free injection may alternatively be used. A typical
intramuscular dose is 0.5 ml.
[0145] The invention may be used to elicit systemic and/or mucosal
immunity, preferably to elicit an enhanced systemic and/or mucosal
immunity.
[0146] Dosage can be by a single dose schedule or a multiple dose
schedule. Multiple doses may be used in a primary immunisation
schedule and/or in a booster immunisation schedule. In a multiple
dose schedule the various doses may be given by the same or
different routes e.g. a parenteral prime and mucosal boost, a
mucosal prime and parenteral boost, etc. Multiple doses will
typically be administered at least 1 week apart (e.g. about 2
weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks,
about 10 weeks, about 12 weeks, about 16 weeks, etc.). In one
embodiment, multiple doses may be administered approximately 6
weeks, 10 weeks and 14 weeks after birth. e.g. at an age of 6
weeks, 10 weeks and 14 weeks, as often used in the World Health
Organisation's Expanded Program on Immunisation ("EPI"). In an
alternative embodiment, two primary doses are administered about
two months apart, e.g. about 7, 8 or 9 weeks apart, followed by one
or more booster doses about 6 months to 1 year after the second
primary dose, e.g. about 6, 8, 10 or 12 months after the second
primary dose. In a further embodiment, three primary doses are
administered about two months apart, e.g. about 7, 8 or 9 weeks
apart, followed by one or more booster doses about 6 months to 1
year after the third primary dose, e.g. about 6, 8, 10, or 12
months after the third primary dose.
[0147] General
[0148] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, immunology and pharmacology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., references 32-38, etc.
[0149] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0150] The term "about" in relation to a numerical value x is
optional and means, for example, x+10%.
[0151] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0152] References to charge, to cations, to anions, to zwitterions,
etc., are taken at pH 7.
[0153] TLR3 is the Toll-like receptor 3. It is a single
membrane-spanning receptor which plays a key role in the innate
immune system. Known TLR3 agonists include poly(I:C). "TLR3" is the
approved HGNC name for the gene encoding this receptor, and its
unique HGNC ID is HGNC:11849. The RefSeq sequence for the human
TLR3 gene is GI:2459625.
[0154] TLR7 is the Toll-like receptor 7. It is a single
membrane-spanning receptor which plays a key role in the innate
immune system. Known TLR7 agonists include e.g. imiquimod. "TLR7"
is the approved HGNC name for the gene encoding this receptor, and
its unique HGNC ID is HGNC:15631. The RefSeq sequence for the human
TLR7 gene is GI:67944638.
[0155] TLR8 is the Toll-like receptor 8. It is a single
membrane-spanning receptor which plays a key role in the innate
immune system. Known TLR8 agonists include e.g. resiquimod. "TLR8"
is the approved HGNC name for the gene encoding this receptor, and
its unique HGNC ID is HGNC:15632. The RefSeq sequence for the human
TLR8 gene is GI:20302165.
[0156] The RIG-1-like receptor ("RLR") family includes various RNA
helicases which play key roles in the innate immune system[39].
RLR-1 (also known as RIG-1 or retinoic acid inducible gene I) has
two caspase recruitment domains near its N-terminus. The approved
HGNC name for the gene encoding the RLR-1 helicase is "DDX58" (for
DEAD (Asp-Glu-Ala-Asp) box polypeptide 58) and the unique HGNC ID
is HGNC:19102. The RefSeq sequence for the human RLR-1 gene is
GI:77732514. RLR-2 (also known as MDA5 or melanoma
differentiation-associated gene 5) also has two caspase recruitment
domains near its N-terminus. The approved HGNC name for the gene
encoding the RLR-2 helicase is "IFIH1" (for interferon induced with
helicase C domain 1) and the unique HGNC ID is HGNC: 18873. The
RefSeq sequence for the human RLR-2 gene is GI: 27886567. RLR-3
(also known as LGP2 or laboratory of genetics and physiology 2) has
no caspase recruitment domains. The approved HGNC name for the gene
encoding the RLR-3 helicase is "DHX58" (for DEXH (Asp-Glu-X-His)
box polypeptide 58) and the unique HGNC ID is HGNC:29517. The
RefSeq sequence for the human RLR-3 gene is GI: 149408121.
[0157] PKR is a double-stranded RNA-dependent protein kinase. It
plays a key role in the innate immune system. "EIF2AK2" (for
eukaryotic translation initiation factor 2-alpha kinase 2) is the
approved HGNC name for the gene encoding this enzyme, and its
unique HGNC ID is HGNC:9437. The RefSeq sequence for the human PKR
gene is GI:208431825.
MODES FOR CARRYING OUT THE INVENTION
[0158] RNA Replicons
[0159] Various replicons are used below. In general these are based
on a hybrid alphavirus genome with non-structural proteins from
venezuelan equine encephalitis virus (VEEV), a packaging signal
from sindbis virus, and a 3' UTR from Sindbis virus or a VEEV
mutant. The replicon is about 10 kb long and has a poly-A tail.
[0160] Plasmid DNA encoding alphavirus replicons (named:
pT7-mVEEV-FL RSVF or A317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or
A50) served as a template for synthesis of RNA in vitro. The
replicons contain the alphavirus genetic elements required for RNA
replication but lack those encoding gene products necessary for
particle assembly, the structural proteins are instead replaced by
a protein of interest (either a reporter, such as SEAP or GFP, or
an immunogen, such as full-length RSV F protein) and so the
replicons are incapable of inducing the generation of infectious
particles. A bacteriophage (T7 or SP6) promoter upstream of the
alphavirus cDNA facilitates the synthesis of the replicon RNA in
vitro and a hepatitis delta virus (HDV) ribozyme immediately
downstream of the poly(A)-tail generates the correct 3'-end through
its self-cleaving activity.
[0161] Following linearization of the plasmid DNA downstream of the
HDV ribozyme with a suitable restriction endonuclease, run-off
transcripts were synthesized in vitro using T7 or SP6 bacteriophage
derived DNA-dependent RNA polymerase. Transcriptions were performed
for 2 hours at 37.degree. C. in the presence of 7.5 mM (T7 RNA
polymerase) or 5 mM (SP6 RNA polymerase) of each of the nucleoside
triphosphates (ATP, CTP, GTP and UTP) following the instructions
provided by the manufacturer (Ambion). Following transcription the
template DNA was digested with TURBO DNase (Ambion). The replicon
RNA was precipitated with LiCl and reconstituted in nuclease-free
water. Uncapped RNA was capped post-transcriptionally with Vaccinia
Capping Enzyme (VCE) using the ScriptCap m7G Capping System
(Epicentre Biotechnologies) as outlined in the user manual;
replicons capped in this way are given the "v" prefix e.g. vA317 is
the A317 replicon capped by VCE. Post-transcriptionally capped RNA
was precipitated with LiCl and reconstituted in nuclease-free
water. The concentration of the RNA samples was determined by
measuring OD.sub.260nm. Integrity of the in vitro transcripts was
confirmed by denaturing agarose gel electrophoresis.
[0162] Liposomal Encapsulation
[0163] RNA was encapsulated in liposomes made by the method of
references 9 and 40. The liposomes were made of 10% DSPC
(zwitterionic), 40% DlinDMA (cationic), 48% cholesterol and 2%
PEG-conjugated DMG (2 kDa PEG). These proportions refer to the %
moles in the total liposome.
[0164] DlinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was
synthesized using the procedure of reference 5. DSPC
(1,2-Diastearoyl-sn-glycero-3-phosphocholine) was purchased from
Genzyme. Cholesterol was obtained from Sigma-Aldrich.
PEG-conjugated DMG
(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol), ammonium salt), DOTAP
(1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and
DC-chol
(3.beta.-[N--(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
hydrochloride) were from Avanti Polar Lipids.
[0165] In general, eight different methods have been used for
preparing liposomes according to the invention. These are referred
to in the text as methods (A) to (H) and they differ mainly in
relation to filtration and TFF steps. Details are as follows:
[0166] (A) Fresh lipid stock solutions in ethanol were prepared. 37
mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg
of PEG DMG 2000 were weighed and dissolved in 7.55 mL of ethanol.
The freshly prepared lipid stock solution was gently rocked at
37.degree. C. for about 15 min to form a homogenous mixture. Then,
755 .mu.L of the stock was added to 1.245 mL ethanol to make a
working lipid stock solution of 2 mL. This amount of lipids was
used to form liposomes with 250 .mu.g RNA. A 2 mL working solution
of RNA was also prepared from a stock solution of .about.1
.mu.g/.mu.L in 100 mM citrate buffer (pH 6). Three 20 mL glass
vials (with stir bars) were rinsed with RNase Away solution
(Molecular BioProducts, San Diego, Calif.) and washed with plenty
of MilliQ water before use to decontaminate the vials of RNases.
One of the vials was used for the RNA working solution and the
others for collecting the lipid and RNA mixes (as described later).
The working lipid and RNA solutions were heated at 37.degree. C.
for 10 min before being loaded into 3 cc luer-lok syringes. 2 mL of
citrate buffer (pH 6) was loaded in another 3 cc syringe. Syringes
containing RNA and the lipids were connected to a T mixer (PEEK.TM.
500 .mu.m ID junction, Idex Health Science, Oak Harbor. Wash.)
using FEP tubing (fluorinated ethylene-propylene; al FEP tubing has
a 2 mm internal diameter.times.3 mm outer diameter, supplied by
Idex Health Science). The outlet from the T mixer was also FEP
tubing. The third syringe containing the citrate buffer was
connected to a separate piece of FEP tubing. All syringes were then
driven at a flow rate of 7 mL/min using a syringe pump. The tube
outlets were positioned to collect the mixtures in a 20 mL glass
vial (while stirring). The stir bar was taken out and the
ethanol/aqueous solution was allowed to equilibrate to room
temperature for 1 hour. 4 ml of the mixture was loaded into a 5 cc
syringe, which was connected to a piece of FEP tubing and in
another 5 cc syringe connected to an equal length of FEP tubing, an
equal amount of 100 mM citrate buffer (pH 6) was loaded. The two
syringes were driven at 7 mL/min flow rate using the syringe pump
and the final mixture collected in a 20 mL glass vial (while
stirring). Next, the mixture collected from the second mixing step
(liposomes) were passed through a Mustang Q membrane (an
anion-exchange support that binds and removes anionic molecules,
obtained from Pall Corporation, AnnArbor, Mich., USA). Before
passing the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL
of 100 mM citrate buffer (pH 6) were successively passed through
the Mustang membrane. Liposomes were warmed for 10 min at
37.degree. C. before passing through the membrane. Next, liposomes
were concentrated to 2 mL and dialyzed against 10-15 volumes of
1.times.PBS using TFF before recovering the final product. The TFF
system and hollow fiber filtration membranes were purchased from
Spectrum Labs and were used according to the manufacturer's
guidelines. Polysulfone hollow fiber filtration membranes (part
number P/N: X1AB-100-20P) with a 100 kD pore size cutoff and 8
cm.sup.2 surface area were used. For in vitro and in vivo
experiments, formulations were diluted to the required RNA
concentration with 1.times.PBS. [0167] (B) As method (A) except
that, after rocking, 226.7 .mu.L of the stock was added to 1.773 mL
ethanol to make a working lipid stock solution of 2 mL, thus
modifying the lipid:RNA ratio. [0168] (C) As method (B) except that
the Mustang filtration was omitted, so liposomes went from the 20
mL glass vial into the TFF dialysis. [0169] (D) As method (C)
except that the TFF used polyethersulfone (PES) hollow fiber
membranes (part number P-C1-100E-100-01N) with a 100 kD pore size
cutoff and 20 cm.sup.2 surface area. [0170] (E) As method (D)
except that a Mustang membrane was used, as in method (A). [0171]
(F) As method (A) except that the Mustang filtration was omitted,
so liposomes went from the 20 mL glass vial into the TFF dialysis.
[0172] (G) As method (D) except that a 4 mL working solution of RNA
was prepared from a stock solution of .about.1 .mu.g/.mu.L in 100
mM citrate buffer (pH 6). Then four 20 mL glass vials were prepared
in the same way. Two of them were used for the RNA working solution
(2 mL in each vial) and the others for collecting the lipid and RNA
mixes, as in (C). Rather than use T mixer, syringes containing RNA
and the lipids were connected to a Mitos Droplet junction Chip (a
glass microfluidic device obtained from Syrris, Part no. 3000158)
using PTFE tubing (0.03 inches internal diameter.times. 1/16 inch
outer diameter) using a 4-way edge connector (Syrris). Two RNA
streams and one lipid stream were driven by syringe pumps and the
mixing of the ethanol and aqueous phase was done at the X junction
(100 .mu.m.times.105 .mu.m) of the chip. The flow rate of all three
streams was kept at 1.5 mL/min, hence the ratio of total aqueous to
ethanolic flow rate was 2:1. The tube outlet was positioned to
collect the mixtures in a 20 mL glass vial (while stirring). The
stir bar was taken out and the ethanol/aqueous solution was allowed
to equilibrate to room temperature for 1 h. Then the mixture was
loaded in a 5 cc syringe, which was fitted to another piece of the
PTFE tubing, in another 5 cc syringe with equal length of PTFE
tubing, an equal volume of 100 mM citrate buffer (pH 6) was loaded.
The two syringes were driven at 3 mL/min flow rate using a syringe
pump and the final mixture collected in a 20 mL glass vial (while
stirring). Next, liposomes were concentrated to 2 mL and dialyzed
against 10-15 volumes of 1.times.PBS using TFF, as in (D). [0173]
(H) As method (A) except that the 2 mL working lipid stock solution
was made by mixing 120.9 .mu.L of the lipid stock with 1.879 mL
ethanol. Also, after mixing in the T mixer the liposomes from the
20 mL vial were loaded into Pierce Slide-A-Lyzer Dialysis Cassette
(Thermo Scientific, extra strength, 0.5-3 mL capacity) and dialyzed
against 400-500 mL of 1.times.PBS overnight at 4.degree. C. in an
autoclaved plastic container before recovering the final
product.
[0174] Methods (A) to (H) as disclosed above use a N:P ratio of 8:1
for a specified amount of RNA. This ratio can readily be varied by
changing the concentration of RNA in the RNA working solution.
[0175] pKa Measurement
[0176] The pKa of a lipid is measured in water at standard
temperature and pressure using the following technique: [0177] 2 mM
solution of lipid in ethanol is prepared by weighing the lipid and
dissolving in ethanol. 0.3 mM solution of fluorescent probe toluene
nitrosulphonic acid (TNS) in ethanol:methanol 9:1 is prepared by
first making 3 mM solution of TNS in methanol and then diluting to
0.3 mM with ethanol. [0178] An aqueous buffer containing sodium
phosphate, sodium citrate sodium acetate and sodium chloride, at
the concentrations 20 mM, 25 mM, 20 mM and 150 mM, respectively, is
prepared. The buffer is split into eight parts and the pH adjusted
either with 12N HCl or 6N NaOH to 4.44-4.52, 5.27, 6.15-6.21, 6.57,
7.10-7.20, 7.72-7.80, 8.27-8.33 and 10.47-11.12, 400 .mu.L of 2 mM
lipid solution and 800 .mu.L of 0.3 mM TNS solution are mixed.
[0179] 7.5 .mu.L of probe/lipid mix are added to 242.5 .mu.L of
buffer in a 1 mL 96 well plate. This is done with all eight
buffers. After mixing, 100 .mu.L of each probe/lipid/buffer mixture
is transferred to a 250 .mu.L black with clear bottom 96 well plate
(e.g. model COSTAR 3904, Corning). A convenient way of performing
this mixing is to use the Tecan Genesis RSP150 high throughput
liquid handler and Gemini Software. [0180] Fluorescence of each
probe/lipid/buffer mixture is measured (e.g. with a SpectraMax M5
spectrophotometer and SoftMax pro 5.2 software) with 322 nm
excitation, 431 nm emission (auto cutoff at 420 nm). [0181] After
the measurement, the background fluorescence value of an empty well
on the 96 well plate is subtracted from each probe/lipid/buffer
mixture. The fluorescence intensity values are then normalized to
the value at lowest pH. The normalized fluorescence intensity is
then plotted against pH and a line of best fit is provided. [0182]
The point on the line of best fit at which the normalized
fluorescence intensity is equal to 0.5 is found. The pH
corresponding to normalized fluorescence intensity equal to 0.5 is
found and is considered the pKa of the lipid.
[0183] This method gives a pKa of 5.8 for DLinDMA, a preferred
cationic lipid for use with the invention.
[0184] Varying the N:P Ratio for Immunogen Delivery
[0185] Self-replicating replicon (vA317) encoding RSV F protein.
BALB/c mice, 4 or 8 animals per group, were given bilateral
intramuscular vaccinations (50 .mu.L per leg) on days 0 and 21 with
the replicon (1 .mu.g) alone or formulated as liposomes with the
RV01, RV05 or RV13. The RV01 liposomes had 40% DlinDMA. 10% DSPC,
48% cholesterol and 2% PEG-DMG. The RV05(01) liposomes had 40%
cationic lipid, 48% cholesterol, 10% DSPC, and 2% PEG-DMG; the
RV05(02) liposomes had 60% cationic lipid, 38% cholesterol, and 2%
PEG-DMG. The RV13 liposomes had 40% DOTAP, 10% DPE, 48% cholesterol
and 2% PEG-DMG. For comparison, naked plasmid DNA (20 .mu.g)
expressing the same RSV-F antigen was delivered either using
electroporation or with RV01(10) liposomes (0.1 g DNA). Four mice
were used as a naive control group.
[0186] These liposomes were prepared by method (D), except for
RV01(05) which used method (B). The RNA concentration was varied to
give different N:P ratios as shown in the table below.
[0187] The Z average particle diameter, polydispersity index and
encapsulation efficiency of the liposomes were as follows, also
showing the N:P ratio:
TABLE-US-00001 RV Zav (nm) pdI % encapsulation N:P ratio RV01 (10)
158.6 0.088 90.7 8:1 RV01 (08) 156.8 0.144 88.6 16:1 RV01 (05)
136.5 0.136 99 8:1 RV01 (09) 153.2 0.067 76.7 4:1 RV05 (01) 148
0.127 80.6 8:1 RV05 (02) 177.2 0.136 72.4 8:1 RV01 (10) 134.7 0.147
87.8 * 8:1 RV13 (02) 128.3 0.179 97 8:1 * For this RV01(10)
formulation the nucleic acid was DNA not RNA
[0188] Serum was collected for antibody analysis on days 14, 36 and
49. Spleens were harvested from mice at day 49 for T cell
analysis.
[0189] F-specific serum IgG titers (GMT) were as follows:
TABLE-US-00002 RV Day 14 Dav 36 Naked DNA plasmid 439 6712 Naked
A317 RNA 78 2291 RV01 (10) 3020 26170 RV01 (08) 2326 9720 RV01 (05)
5352 54907 RV01 (09) 4428 51316 RV05 (01) 1356 5346 RV05 (02) 961
6915 RV01 (10) DNA 5 13 RV13 (02) 644 3616
[0190] The proportion of T cells which are cytokine-positive and
specific for RSV F51-66 peptide are as follows, showing only
figures which are statistically significantly above zero:
TABLE-US-00003 CD4+CD8- CD4-CD8+ RV IFN.gamma. IL2 IL5 TNF.alpha.
IFN.gamma. IL2 IL5 TNF.alpha. Naked DNA plasmid 0.04 0.07 0.10 0.57
0.29 0.66 Naked A317 RNA 0.04 0.05 0.08 0.57 0.23 0.67 RV01 (10)
0.07 0.10 0.13 1.30 0.59 1.32 RV01 (08) 0.02 0.04 0.06 0.46 0.30
0.51 RV01 (05) 0.08 0.12 0.15 1.90 0.68 1.94 RV01 (09) 0.06 0.08
0.09 1.62 0.67 1.71 RV05 (01) 0.06 0.04 0.19 RV05 (02) 0.05 0.07
0.11 0.64 0.35 0.69 RV01 (10) DNA 0.03 0.08 RV13 (02) 0.03 0.04
0.06 1.15 0.41 1.18
[0191] Thus the liposome formulations significantly enhanced
immunogenicity relative to the naked RNA controls, as determined by
increased F-specific IgG titers and T cell frequencies. Plasmid DNA
formulated with liposomes, or delivered naked using
electroporation, was significantly less immunogenic than
liposome-formulated self-replicating RNA.
[0192] The RV01 and RV05 RNA vaccines were more immunogenic than
the RV13 (DOTAP) vaccine. These formulations had comparable
physical characteristics and were formulated with the same
self-replicating RNA, but they contain different cationic lipids.
RV01 and RV05 both have a tertiary amine in the headgroup with a
pKa of about 5.8, and also include unsaturated alkyl tails. RV13
has unsaturated alkyl tails but its headgroup has a quaternary
amine and is very strongly cationic. These results suggest that
lipids with tertiary amines with pKas in the range 5.0 to 7.6 are
superior to lipids such as DOTAP, which are strongly cationic, when
used in a liposome delivery system for RNA.
[0193] The N:P ratio had an impact on immunogenicity, with 4:1
(RV01(09))>8:1 (RV01(10))>16:1 (RV01(08)).
[0194] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
TABLE-US-00004 TABLE 1 useful phospholipids DDPC
1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA
1,2-Dierucoyl-sn-Glycero-3-Phosphate DEPC
1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE
1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG
1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .)
DLOPC 1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA
1,2-Dilauroyl-sn-Glycero-3-Phosphate DLPC
1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE
1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine DLPG
1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DLPS
1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine DMG
1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine DMPA
1,2-Dimyristoyl-sn-Glycero-3-Phosphate DMPC
1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE
1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine DMPG
1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DMPS
1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine DOPA
1,2-Dioleoyl-sn-Glycero-3-Phosphate DOPC
1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE
1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine DOPG
1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DOPS
1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine DPPA
1,2-Dipalmitoyl-sn-Glycero-3-Phosphate DPPC
1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE
1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine DPPG
1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .)
DPPS 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine DPyPE
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA
1,2-Distearoyl-sn-Glycero-3-Phosphate DSPC
1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPE
1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanolamine DSPG
1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .)
DSPS 1,2-Distearoyl-sn-Glycero-3-phosphatidylserine EPC Egg-PC HEPC
Hydrogenated Egg PC HSPC High purity Hydrogenated Soy PC HSPC
Hydrogenated Soy PC LYSOPC MYRISTIC
1-Myristoyl-sn-Glycero-3-phosphatidylcholine LYSOPC PALMITIC
1-Palmitoyl-sn-Glycero-3-phosphatidylcholine LYSOPC STEARIC
1-Stearoyl-sn-Glycero-3-phosphatidylcholine Milk Sphingomyelin MPPC
1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidylcholine MSPC
1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine PMPC
1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine POPC
1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine POPE
1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidylethanolamine POPG
1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol) . . .] PSPC
1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine SMPC
1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidyicholine SOPC
1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine SPPC
1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine
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